Establishment and characterization of a human cell line, HS-MM, derived from a case of clear cell sarcoma

The characteristics of a new human clear cell sarcoma (CCSa) cell line, HS-MM, established from the pleural effusion in a 39-year-old man with lung metastasis, have been morphologically studied in vitro and in vivo. HS-MM cells growing on a cover-slip were round or spindle in shape with round nuclei containing extremely prominent nucleoli. Heterotransplantation of the cells into nude mice was easily succeeded following tumor development. Light microscopically, HS-MM cells, both in vitro and in vivo, were positive for anti-S-100 protein and anti-melanoma specific antibodies with immunostain, but no melanin pigment was detected in them. Ultrastructurally, the cells had round euchromatin-rich nuclei with large nucleoli revealing conspicuous nucleolonema, and contained a few mitochondria, rough endoplasmic reticulum and lysosomal dense bodies, besides a large amount of glycogen, but no melanosome in their cytoplasm. HS-MM cells retained and fully expressed morphologically unique characteristics as a CCSa, compatible with amelanotic type of malignant melanoma also. This cell line, HS-MM, therefore, proves to be extremely useful for clinicopathological studies on a CCSa.     

Mitochondrial respiration of complex II is not lower than that of complex I in mouse skeletal muscle

Skeletal muscle (SKM) requires a large amount of energy, which is produced mainly by mitochondria, for their daily functioning. Of the several mitochondrial complexes, it has been reported that the dysfunction of complex II is associated with several diseases, including myopathy. However, the degree to which complex II contributes to ATP production by mitochondria remains unknown. As complex II is not included in supercomplexes, which are formed to produce ATP efficiently, we hypothesized that complex II-linked respiration was lower than that of complex I. In addition, differences in the characteristics of complex I and II activity suggest that different factors might regulate their function. The isolated mitochondria from gastrocnemius muscle was used for mitochondrial respiration measurement and immunoblotting in male C57BL/6J mice. Student paired t-tests were performed to compare means between two groups. A univariate linear regression model was used to determine the correlation between mitochondrial respiration and proteins. Contrary to our hypothesis, complex II-linked respiration was not significantly less than complex I-linked respiration in SKM mitochondria (complex I vs complex II, 3402 vs 2840 pmol/[s × mg]). Complex I-linked respiration correlated with the amount of complex I incorporated in supercomplexes (r = 0.727, p < 0.05), but not with the total amount of complex I subunits. In contrast, complex II-linked respiration correlated with the total amount of complex II (r = 0.883, p < 0.05), but not with the amount of each complex II subunit. We conclude that both complex I and II play important roles in mitochondrial respiration and that the assembly of both supercomplexes and complex II is essential for the normal functioning of complex I and II in mouse SKM mitochondria.     

The Structures of Myocotrienins I. and II,a Novel Class of Ansamycin Antibiotic

Endopolygalacturonase I [EC 3.2.1.15], the major component of endopolygalacturonases causing silver-leaf symptoms, was purified from culture liquids of Stereum purpureum by column chromatographies on CM-52 and Sephadex G-100. The purified enzyme was homogeneous on Polyacrylamide gel electrophoresis and ultracentrifugation. The sedimentation coefficient (S_20,W) was determined to be 3.21 S, and the molecular weight was estimated to be 40,000 by gel filtration, 41,000 by SDS-polyacrylamide gel electrophoresis and 44,000 by sedimentation equilibrium. The enzyme had an isoelectric point of pH 8.5. The optimal pH of the enzyme was 3.5 for trigalacturonic acid, 4.0 for tetragalacturonic acid, and 4.5 for pectic acid. The enzyme was stable in the range of pH 4.0 to 9.0 and up to 70%C for 30 min. The amount of the enzyme which was required to induce silver-leaf symptoms on apple trees was 20 μg/tree.     

Chromosome aberrations, micronuclei and sister-chromatid exchanges (SCEs) in rat liver induced in vivo by hepatocarcinogens including heterocyclic amines

The induction of chromosome aberrations, micronuclei and SCEs was studied in hepatocytes of F344 rats exposed in vivo to hepatocarcinogens. Hepatocytes were isolated and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after a culture period of 48 h. Oral administration of dimethylnitrosamine at doses of 2.5-20 mg/kg body weight (bw) induced (1) chromosome aberrations in up to 27% of the metaphase cells 2-48 h after its administration, (2) SCEs with a frequency of up to 0.9 per chromosome 2-48 h after its administration, and (3) micronuclei in up to 2.9% of the cells 16-48 h after its administration. Oral administration of 2-acetylaminofluorene at doses of 6.25-200 mg/kg bw induced (1) chromosome aberrations in up to 35% of the metaphase cells after 2-48 h, (2) SCEs at up to 0.9 per chromosome and (3) micronuclei in up to 2.5% of the cells with a maximum after 4 h. Oral administration of CCl4, a non-genotoxic hepatocarcinogen, at a dose of 1600 mg/kg bw did not induce chromosome aberrations, SCEs or micronuclei within 4-72 h. Intraperitoneal injections of Trp-P-1, Glu-P-1, MeIQx, IQ and nitro-IQ resulted in chromosome aberrations in up to 16% of the metaphase cells and SCEs at up to 0.9 per chromosome, while injections of Trp-P-2 and Glu-P-2 produced SCEs at up to 0.7 and 1.1 per chromosome, respectively. The present method of in vivo cytogenetic assay using rats without partial hepatectomy or mitogen treatment in vivo should be useful for evaluating the tumor-initiating activities of hepatocarcinogens.     

Alkaline elution of DNA from stomach pyloric mucosa of rats treated with glyoxal

DNA damage in the pyloric mucosa of the stomach of male F344 rats was determined by the alkaline elution method after administration of glyoxal, a direct-acting mutagen present in various heated foods, by gastric intubation. Glyoxal at doses of 50-550 mg/kg body weight induced DNA damage in the pyloric mucosa of rat stomach, detected by a 5- to 12-fold increase in the elution rate constant 2 h after its administration. N-Methyl-N'-nitro-N-nitrosoguanidine, a glandular stomach carcinogen, used as a positive control at doses of 1-100 mg/kg body weight induced a 11- to 24-fold increase in the elution rate constant, while 2-acetylaminofluorene, which is not a gastric carcinogen, given as a negative control at doses of 200-400 mg/kg body weight did not increase the elution rate constant. Thus glyoxal, which was previously suggested to induce unscheduled DNA synthesis in the pyloric mucosa of rat stomach, was confirmed to be genotoxic in this region.     

Genotoxicity and cell proliferative activity of a nitrosated Oroxylum indicum Vent fraction in the pyloric mucosa of rat stomach.

In vivo genotoxic activity and cell proliferative activity were examined in the stomach mucosa of male F344 rats by in vivo short-term methods after oral administration of a nitrosated Oroxylum indicum Vent (OiV) fraction, which had been found to be mutagenic without S9 mix to Salmonella typhimurium TA98 and TA100. Administration of the nitrosated OiV fraction at doses of 1 and 2 g/kg body weight induced dose-dependent DNA single-strand scission (p less than 0.02), determined by the alkaline elution method, in the stomach pyloric mucosa 2 h after its administration: a dose of 2 g/kg body weight induced an 18-fold increase in the DNA elution rate constant. Administration of the nitrosated OiV fraction at doses of 0.7-2.8 g/kg body weight also induced dose-dependent increases, up to 11-fold (p less than 0.05), in replicative DNA synthesis in the stomach pyloric mucosa 16 h after its administration. Moreover administration of the nitrosated OiV fraction at doses of 0.25-2.0 g/kg body weight induced dose-dependent increases, up to 100-fold, in ornithine decarboxylase activity in the stomach pyloric mucosa with a maximum 4 h after its administration. These results demonstrate that the nitrosated OiV fraction has genotoxic and cell proliferative activity in the pyloric mucosa of rat stomach in vivo.     

Premature induction of pepsinogen in developing rat gastric mucosa by hormones

Daily injection of ACTH into rats from the age of 9 days caused precocious development of peptic activity in the gastric mucosa. A single injection of hydrocortisone into rats aged 2 to 9 days also evoked premature development of peptic activity in the gastric mucosa. The patterns on polyacrylamide gel electrophoresis of pepsinogen from adults and from infants treated with hormone were quite similar but differed from that of untreated infants. Enzyme from the gastric mucosa of adult rats and enzyme evoked with hormones were more stable than that from infant rats.     

Patterns of Genomic Integration of Nuclear Chloroplast DNA Fragments in Plant Species

The transfer of organelle DNA fragments to the nuclear genome is frequently observed in eukaryotes. These transfers are thought to play an important role in gene and genome evolution of eukaryotes. In plants, such transfers occur from plastid to nuclear [nuclear plastid DNAs (NUPTs)] and mitochondrial to nuclear (nuclear mitochondrial DNAs) genomes. The amount and genomic organization of organelle DNA fragments have been studied in model plant species, such as Arabidopsis thaliana and rice. At present, publicly available genomic data can be used to conduct such studies in non-model plants. In this study, we analysed the amount and genomic organization of NUPTs in 17 plant species for which genome sequences are available. The amount and distribution of NUPTs varied among the species. We also estimated the distribution of NUPTs according to the time of integration (relative age) by conducting sequence similarity analysis between NUPTs and the plastid genome. The age distributions suggested that the present genomic constitutions of NUPTs could be explained by the combination of the rapidly eliminated deleterious parts and few but constantly existing less deleterious parts.